Leishmaniasis is a chronic protozoal disease of man associated with both protective and immunopathologic immune responses. To define these responses, a vaccine against Leishmania major was developed in which protective immunity is induced in BALB/c mice by intraperitoneal immunization with a soluble fraction of promastigotes. To identify functional immunogens, the soluble antigen preparation was passed over an FPLC Mono Q column, and separated into 9 pools. We then assessed T cell responses, including lymphocyte transformation, IL-2 production, delayed hypersensitivity and macrophage activating factor (MAF) production, as well as antibody responses, to each pool. Although several pools were capable of stimulating T cells only one pool induced significant protection. The protective pool contains a group of highly negatively charged molecules, including both proteins and DNA. Enzymatic removal of protein destroyed the protective activity of the fraction, while digestion of DNA had no effect. Several proteins were unique to this pool, including a predominant 35Kd antigen. Monoclonal antibodies have been raised against these unique molecules and will be used to affinity purify potentially protective antigens. These results suggest that only a small subset of the T cells stimulated by leishmanial antigens induce protection. We are presently investigating the possibility that the non-protective T cells may induce immunopathologic responses. Finally, we have studied non-healing L. major infections in two other mouse models. The P/J mouse strain fails to heal following infection, and it has been suggested that the failure to heal is due to a qualitative defect in MAF production. We overcame this defect by vaccination, as well as in vivo removal of a subset of regulatory T cells, suggesting that the defect in MAF production is due to a regulatory T cell imbalance. We have also shown that B cell deficient C3H/HeN mice fail to heal normally, and found that this may be due to the lack of a T cell subset that requires B cells for its normal maturation.